JP6918808B2 - Antibody measurement method using antigen-supporting insoluble carrier particles in which antigens are immobilized by different methods, reagents for antibody measurement - Google Patents

Description

The present invention relates to a method for measuring a biological substance, and more specifically, an antibody measuring means for using an antigen-supporting insoluble carrier particle in which an antigen is immobilized by a different method, for example, an antigen-supporting latex particle, which are mixed with each other. Is an invention related to.

Currently, in diagnostic agents using insoluble carrier particles, reagents using the latex agglutination method as one of immunological measurement methods are widely used.

The latex agglutination method, using latex particles supporting an antigen or antibody in a liquid phase, to form a measuring system for detecting the antibody or antigen. Based on the property that latex particles aggregate due to the formation of immune complexes, the degree of aggregation can be visually confirmed, or the increase in turbidity can be measured optically as a change in absorbance or scattered light intensity (hereinafter,). Also referred to as latex method: Patent Document 1).

The sandwich immunoassay method such as ELISA has an accurate measurement itself and can be used as a reference method, but in the process of measurement, components other than the substance to be measured and components in the measurement reagent contained in the measurement sample are removed. Since it includes a cleaning step, it takes a long time for measurement and has a drawback that the measurement operation is complicated. Therefore, it is not suitable for measuring a large number of measurement samples in a short time.

On the other hand, the latex method is easy to operate, easy to apply to general-purpose automatic biochemical analyzers widely used in clinical tests, and enables measurement in a short time and measurement of a large number of test samples. Is.

Therefore, the latex method is used in many clinical examination items.

Japanese Unexamined Patent Publication No. 53-62826

An object of the present invention is to expand the measurement range in antibody measurement using insoluble carrier particles such as latex particles carrying an antigen, and to improve sensitivity and specificity. In particular, it provides a means for obtaining good measurement results even when a mixture of a plurality of substances such as a lysate derived from a microorganism is supported.

When developing a diagnostic agent using insoluble carrier particles, the molecular weight of the substance to be supported on the insoluble carrier particles and the selection of the supporting method have a great influence on the performance.

For example, when a physical adsorption method is used, that is, when a protein or the like is supported on the insoluble carrier particles by direct contact using an electrostatic interaction (hydrophobic interaction) between the insoluble carrier particles and the supported protein. Is suitable for carrying relatively high molecular weight substances. However, carrying a low molecular weight substance or hapten has a drawback that it may be buried on the surface of an insoluble carrier or buried by a blocking agent used for suppressing non-specific aggregation. In addition, the physical adsorption method has some drawbacks in supporting proteins with very few hydrophobic amino acid residues.

When the chemical bonding method is used, that is, a method of covalently bonding a carboxyl group and an amino group on the surface of an insoluble carrier particle or a protein with a coupling reagent such as carbodiimide, an aldehyde group or a tosyl group on the surface of an insoluble carrier particle and an amino group of the protein. When a protein or the like is supported on the insoluble carrier particles by a method of binding with or the like, it is relatively easy to support a low molecular weight protein or hapten, which is difficult for the physical adsorption method. Furthermore, when a chemical bond method is used, the functional group of the insoluble carrier is appropriately selected to control the binding site on the protein side that forms a covalent bond, that is, the binding site on the protein side (covalently bonded amino acid residue). It is possible to select. However, the problem that it is difficult to support a protein lacking in functional groups (in other words, a protein having high hydrophobicity) is recognized, and further, the process of producing the supported particles is complicated, and the problem of denaturation of the supported protein is also recognized. Be done.

In particular, when supporting a "mixture of multiple substances" such as a lysate derived from a microorganism, if the supporting method is only one of the physical adsorption method and the chemical bonding method, the size and protein of each substance may be changed. Since the contents of hydrophilic amino acid residues and hydrophobic amino acid residues are different, the substances supported are biased, and as a result, good measurement results can be obtained with the obtained antigen-supporting insoluble carrier particles. That is, the type of target antibody to be captured is limited to those that bind to a limited substance, and data on microorganisms that cause diseases, etc. from which the target antibody is derived. There is a problem that the acquisition may not be performed sufficiently.

Further, since an antibody against a microorganism in a living body exists not as an antibody against a single antigen but as a plurality of antibodies against a plurality of antigens, if these plurality of antibodies can be captured at one time, more reliable data acquisition will be achieved. As far as the present inventor knows, there is no example in which insoluble carrier particles carrying a mixture of an unspecified number of substances such as an antigenic substance derived from a living organism are used as an antigen.

The present inventors have investigated the above-mentioned problems, and have investigated the insoluble carrier particles in which the antigenic substance is supported by the physical adsorption method and the insoluble carrier particles in which the antigenic substance is supported by the chemical binding method. By using a solution containing insoluble carrier particles containing both in antibody measurement, high sensitivity and specificity can be achieved with a wide measurement range, especially a mixture of multiple substances such as antigenic substances derived from living organisms. The present invention has been completed by finding that it can be realized even in the case of.

That is, in the present invention, insoluble carrier particles carrying a predetermined antigen by physical adsorption (hereinafter, also referred to as physically adsorbed particles) and insoluble carrier particles carrying the antigen by chemical binding (hereinafter, also referred to as chemically bound particles). ), The solution containing the insoluble carrier particles containing both of () and (hereinafter, also referred to as the solution of the present invention) was brought into contact with a sample separated from a living body capable of containing the target antibody and supported on the carrier particles. Provided is an antibody measuring method (hereinafter, also referred to as the measuring method of the present invention), which comprises detecting the aggregation reaction of the carrier particles due to the antigen-antibody reaction between the antigen and the target antibody in the sample. In the antibody measuring method of the present invention, a diluted solution is preferably used in addition to the containing solution of the present invention. When a diluent is used, it is preferable to dilute the sample separated from the living body that can contain the target antibody with the diluent and then bring it into contact with the solution containing the insoluble carrier particles.

Further, the present invention is a reagent for measuring an antibody (hereinafter referred to as the present invention), which is a liquid containing insoluble carrier particles containing both physically adsorbed particles and chemically bound particles (the liquid contained in the present invention). Also referred to as a measurement reagent of the invention).
Furthermore, the present invention provides a measurement kit (hereinafter, also referred to as the kit of the present invention), which comprises the measurement reagent of the present invention (containing liquid of the present invention) and a diluent.

It is preferable that both the physically adsorbed particles and the chemically bonded particles are provided as latex particles. In addition, insoluble carrier particles such as silica colloid, magnetic particles, and metal colloid can be used. The content liquid is not particularly limited as long as it contains insoluble carrier particles. That is, the contained liquid includes a dispersion liquid, an emulsion liquid, a suspension liquid, a precipitation liquid, a multilayer separation liquid and the like. Further, the insoluble carrier particles in the contained liquid may be subjected to surface treatment as required.

The physically adsorbed particles are insoluble carrier particles in which a predetermined antigen is carried by a physical adsorption method as specified above. The average particle size of the physically adsorbed particles can be used in the range of 0.01 μm to 1.0 μm, preferably 0.05 μm to 0.35 μm, more preferably 0.10 μm to 0.35 μm, and most preferably. It is preferably 0.20 μm −0.35 μm. The physically adsorbed particles can be produced by bringing the antigen to be adsorbed into contact with the solution containing the insoluble carrier particles to be subjected to the physical adsorption method.

The chemically bonded particles are insoluble carrier particles in which a predetermined antigen is supported by a chemical bonding method as specified above. The average particle size of the chemically bonded particles can be used in the range of 0.01 μm to 1.0 μm, preferably 0.05 μm to 0.35 μm, more preferably 0.10 μm to 0.35 μm, and most preferably. It is preferably 0.20 μm −0.35 μm. The chemically bonded particles can be produced by performing an operation of covalently binding the target antigen to the containing liquid of the insoluble carrier particles to be subjected to the chemical bonding method.

That is, the containing liquid of the present invention can be preferably prepared by mixing the above-mentioned "containing liquid of physically adsorbed particles" and "containing liquid of chemically bonded particles".

The mixing ratio (mass ratio) of the physically adsorbed particles and the chemically bonded particles is preferably 10: 1-1: 10 (physisorbed particles: chemically bonded particles), and more preferably 5: 1-1: 5 (physisorbed). Particles: chemically bonded particles), very preferably 3: 1-1: 3 (physisorbed particles: chemically bonded particles), most preferably 3: 1-1: 2 (physisorbed particles: chemically bonded particles). be. However, when the content of physically adsorbed particles in the measurement reagent when measuring the target antibody in the sample or in the mixed solution of the measurement reagent and the diluent is 0.045% by mass or more when a diluent is used. If there is, there is a tendency that the improvement of the effect corresponding to the increase in the content of the particles is not recognized.

The "predetermined" in the "predetermined antigen" can be paraphrased as, for example, "selected". It means "defined" or "selected" as an antigen that binds to the antibody selected for measurement, and once defined or once selected, the antigen is unique. Determine.

The antigen that can be a "predetermined antigen" is not particularly limited and can be widely selected as long as it is a substance that binds to the antibody to be measured, but it is preferably an antigen that binds to the antibody in the sample separated from the living body. That is, it is preferable that an antibody against a substance existing in the living body due to a specific disease or constitution is used as a target antibody, and an antigen that binds to the target antibody is used as a “predetermined antigen”. A typical substance that can be a "predetermined antigen" is a protein, but other substances such as sugar chains and lipids are also included.

In addition, "living body" broadly refers to the body of living things in general, but is usually the human body.

By using the antigen as a "mixture of a plurality of substances", the effects of the present invention are suitably exhibited. Examples of the mixture include antigenic substances derived from living organisms, and preferred examples thereof include lysates derived from microorganisms such as bacteria and viruses.

Further, the effect of the present invention is preferably exhibited when the antigen contains at least one substance having a molecular weight of 5,000 or more, particularly preferably 10,000 or more. By using both the physically adsorbed particles and the chemically bonded particles in combination with respect to the lysate derived from the microorganism, it is possible to present various epitopes as a whole. The upper limit of the molecular weight is not particularly limited as long as it can be supported on the insoluble carrier particles, but it is preferably about 2000000 or less. In some cases, the above-mentioned "mixture of multiple substances" meets the conditions of this specific molecular weight.

The above-mentioned containing solution of the present invention is brought into contact with a sample separated from a living body that can contain a target antibody, and an antigen-antibody between the antigen carried on the insoluble carrier particles of the containing solution and the target antibody in the sample. The degree of agglutination of the carrier particles due to the reaction can be detected.

The "sample isolated from the living body that can contain the target antibody" is not particularly limited as long as it may contain the antibody to be measured, and for example, blood, serum, plasma, urine, lymph, puncture fluid , and the like. Examples include cerebrospinal fluid, sweat, saliva, gastric fluid, lung lavage fluid, feces and the like. Of these, blood, serum and plasma are preferable.

The main "reaction of insoluble carrier particles carrying a predetermined antigen by an antigen-antibody reaction" is an agglutination reaction, which is insoluble using a slide agglutination method, an optical measurement method, a microtiter method, a filter separation method, or the like. By detecting the aggregation of the carrier particles, the desired antibody can be measured.

The measuring method of the present invention is preferably carried out using an automatic biochemical analyzer. The biochemical automatic analyzer is based on an optical measurement method, and usually, at the time of use, a sample such as serum is diluted with a diluent and heated, and then a reagent (in the case of the present invention) is used. , The content of the present invention) is added for measurement. Examples of the biochemical automatic analyzers currently provided include Hitachi 7180, LABOPSECT 008, LABOPSECT 006 (manufactured by Hitachi High-Technologies Co., Ltd.); JCA-BM6010, JCA-BM6050, JCA-BM9130, etc. (JEOL Ltd.) Co., Ltd.); TBA-c16000, TBA-2000FR, TBA-120FR, etc. (manufactured by Toshiba Medical Systems Co., Ltd.); AU680, AU5800, etc. (manufactured by Beckman Coulter Ltd.), etc., but are limited thereto. It's not a thing.

INDUSTRIAL APPLICABILITY The present invention provides a means for measuring an antibody with high sensitivity and specificity, as well as a wide measurement range.

It is a figure which examined the effect on the reactivity of the mixture of the chemically bonded latex particle of the particle diameter 0.145 μm, and the physically adsorbed latex particle of the particle diameter 0.235 μm. It is a figure which examined the effect on the reactivity by changing the mixing amount of the chemically bonded latex particles of a particle size of 0.300 μm with respect to the physically adsorbed latex particles of a particle size of 0.235 μm.

1. 1. The liquid contained in the present invention As described above, the liquid contained in the present invention is a liquid containing both physically adsorbed particles and chemically bonded particles.

(1) Procurement of Insoluble Carrier Particles As described above, it is preferable that both the physically adsorbed particles and the chemically bonded particles are provided as latex particles. Latex is an emulsion in which polymer fine particles (latex particles) are stably dispersed in an aqueous solvent.

The latex can be produced by using a conventional method such as an emulsion polymerization method, a soap-free emulsion polymerization method, a seed polymerization method, a stage feed emulsion polymerization method, a power feed polymerization method, or a suspension polymerization method. The particle size can also be adjusted using conventional methods in each of these latex preparation methods. For example, in the case of the emulsion polymerization method, the particle size of the latex can be adjusted by adjusting the type and amount of the monomer, emulsifier, and initiator, the polymerization temperature, and the like. Of course, it is also possible to use a commercially available latex product.

The type of latex is not particularly limited as long as the physical adsorption method used for producing physically adsorbed particles or the chemical bond method used for producing chemically bonded particles can be applied, and the physical adsorption method and chemical bonding are not particularly limited. It is preferable to select the type suitable for each of the methods.

Examples of latex suitable for the physical adsorption method include polystyrene latex, ultra-low carboxylic acid latex, and hydrophilic group localized latex.

Examples of latex suitable for the chemical bonding method include latex containing latex particles having a carboxyl group, a hydroxyl group, an amino group, an aldehyde group, a tosyl group, or the like on the surface.

It is also possible to use a colored latex that has been colored or a fluorescent latex that has been subjected to a fluorescent substance, whether it is a latex suitable for the physical adsorption method or a latex suitable for the chemical bonding method. ..

As described above, the particle size of the latex particles that perform physical adsorption and the latex particles that perform chemical bonding are not particularly limited, and can be used in the range of 0.01 μm to 1.0 μm, preferably 0.05 μm. It is −0.35 μm, more preferably 0.10 μm −0.35 μm, and most preferably 0.20 μm −0.35 μm.

For each of these two carrying latex particles, the particle size of the latex particles used in the measurement can be determined in order to obtain appropriate sensitivity, specificity and measurement range according to the properties of the carrying antigen and the measurement antibody. .. Specifically, when it is desired to enhance the cohesiveness of latex by an immune reaction, it is preferable to select a medium to large particle size (0.20 to 0.35 μm), but the measurement target (antibody) If the concentration is very high, it may be preferable to select a small particle size (0.10 μm or less).

As described above, examples of the insoluble carrier particles other than the latex particles include silica colloidal particles, magnetic particles, and metal colloidal particles. These insoluble carrier particles can be made into insoluble carrier particles that can be used in the present invention by modifying the particle surface to which a functional group suitable for physical adsorption or chemical bonding is added.

(2) Step of supporting on insoluble carrier particles (a) Supported antigen The antigen to be supported on the insoluble carrier particles is not limited as long as it can bind to the antibody to be measured, but as described above, "plurality". The effect of the present invention is preferably exhibited by using a "mixture of substances" or a "containing substance having a large molecular weight". Examples of the mixture include antigenic substances derived from living organisms, and preferred examples thereof include lysates derived from microorganisms such as bacteria and viruses. For example, the lysates of Helicobacter pylori used in the examples described later include urease B, urease A, Cag A, flagellin, heat shock protein, and the like. Other substances, are contained. Most of the substances contained in the lysates of Helicobacter pylori have a molecular weight of about 10,000 to 200,000.

In order to identify an infectious microorganism in the body such as Helicobacter pylori, a substance peculiar to the infectious microorganism can be used as a supporting antigen. As microorganisms, in addition to this helicobacter pylori, bacteria such as group A lytic bacterium Lensa bacterium, group B lytic bacterium Lensa bacterium, pneumonia bacterium urinary antigen, Escherichia coli O157, Clostridium difficile, Regionella bacterium, cholera bacterium, and meningitis-causing bacterium. Influenza virus, parainfluenza virus, rotavirus, norovirus, Japanese encephalitis virus, mad dog disease virus, poliovirus, echo virus, coxsackie virus A group, coxsackie virus B group, mumps virus, simple herpes virus, varicella / herpes zoster virus, measles virus, Eczema virus, EB virus, papillomavirus, infectious soft tumor virus, limb mouth disease virus, acute hemorrhagic conjunctivitis virus, HAV, HBV, HCV, epidemic keratoconjunctivitis virus, hunter virus, human T lymphocyte-loving virus, Viruses such as HIV, lymphocytic choriomyelitis virus, RS virus, adenovirus, leovirus, rhinovirus, coronavirus; Fungi that cause deep-seated mycosis; liquettia that causes tsutsugamushi disease, erythema fever, rash typhoid, etc .; clazimia, protozoa, parasites and the like.

In addition to microbial-derived antigens, as supporting antigens that can be used in the present invention, antigens against anti-receptor antibodies such as anti-TSH receptor antibody, anti-acetylcholine receptor antibody, and anti-insulin receptor antibody; anti-thyroglobulin antibody, anti-microsome. Antibodies to anti-thyroid antibodies such as antibodies; Anti-pancreatic islet cells antibodies, anti-GAD antibodies, anti-pancreatic islets antibodies such as anti-insulin antibodies; Examples thereof include antigens for organ-specific autoantibodies such as factor antibodies, anti-horizontal muscle antibodies, anti-myocardial antibodies, anti-skin desmograin antibodies, anti-neutrophil cytoplasmic antibodies, and anti-phospholipid antibodies.

Furthermore, as carrying antigens that can be used in the present invention, autoantigens distributed in chromatin such as DNA, nucleosome, histone, polyADP ribose, centromea, Scl-70, Ku; U1RNP, Sm, SS-B / LA, SS -Autoantigens distributed in the nucleoplasm such as A / Ro, PCNA, Ki; Autoantigens distributed in the nucleolus such as U3RNP, Th / To, PM-Sci, RNA polymerase; AA-A / Ro, Jo-1 , Ribosome P, mitochondria M2, antigens against organ-nonspecific autoantigens such as autoantigens distributed in the nucleoplasm such as signal recognition particles.

The above description is an example list, and other antigens applicable at present time can be used, and antigens provided in the future can also be used.

(B) Antigen carrying step The antigen can be supported on the insoluble carrier particles according to a physical adsorption method and a chemical binding method, respectively.

In the physical adsorption method, for example, a solution containing insoluble carrier particles in distilled water or PBS in an amount of about 0.1-1% by mass is mixed with an antigen to be supported, and the insoluble carrier particles are made to support the antigen. Can be done. Further, the unadsorbed antigen and the antigen-supporting carrier particles are separated by centrifugation and redispersed in an aqueous solvent (usually a buffer solution) to prepare a solution containing the physically adsorbed particles. Examples of the buffer solution include Good's buffer solution such as HEPES buffer solution, TRIS buffer solution, glycine buffer solution, boric acid buffer solution and the like.

The chemical bond method utilizes a functional group on the surface of insoluble carrier particles to covalently bond a predetermined antigen. For example, when the functional groups on the surface of the insoluble carrier particles are a carboxyl group, a hydroxyl group, and an amino group, the antigen can be covalently bonded to the surface of the insoluble carrier particles by using carbodiimide, bromcian, and glutaraldehyde as activators, respectively. It is possible. It is also possible to activate the functional groups on the surface of the insoluble carrier particles with an activator in advance and then contact the antigen, or the activator and the antigen may be contacted with the insoluble carrier particles at the same time. When the functional group on the surface of the insoluble carrier particles is an aldehyde group or a tosyl group, it is not necessary to use an activator. Further, it is also possible to insert a spacer molecule such as an oligoamino acid or an aminocarboxylic acid between the insoluble carrier particles and the antigen to carry out a covalent bond.

The above chemical bond reaction is carried out in an aqueous solvent (usually a buffer), and the obtained chemical bond particles are redispersed in an aqueous solvent (usually a buffer) to contain the chemical bond particles. Can be produced. Examples of the buffer solution include Good's buffer solution such as HEPES buffer solution, TRIS buffer solution, glycine buffer solution, boric acid buffer solution and the like.

In this way, physically adsorbed particles and chemically bonded particles can be produced. The physically adsorbed particles and the chemically bonded particles are stored in an aqueous solvent as a liquid containing the insoluble carrier particles.

(3) Containing liquid of the present invention The contained liquid of the present invention is a containing liquid containing both physically adsorbed particles and chemically bonded particles, and the solvent thereof is usually a buffer solution. Examples of the buffer solution include Good's buffer solution such as HEPES buffer solution, TRIS buffer solution, glycine buffer solution, boric acid buffer solution and the like. For example, the containing liquid of the present invention is an aqueous solvent (usually, a buffer) containing the physically adsorbed particles prepared as the containing liquid of the above physically adsorbing particles and the chemically bonded particles prepared as the containing liquid of the chemically bonded particles. It can be produced by mixing in. The method for producing the containing liquid of the present invention is not limited to this as long as a containing liquid containing both physically adsorbed particles and chemically bonded particles can be obtained.

If necessary, the liquid containing the present invention may contain, for example, various electrolytes, stabilizers, surfactants, sensitizers, and the like.

The content ratio of each antigen-carrying carrier particle in the containing liquid of the present invention varies depending on the desired measurement range and sensitivity, but is preferably 10: 1-1: 10 (physically adsorbed particles: chemical bond) in terms of mass ratio. Particles), more preferably 5: 1-1: 5 (physically adsorbed particles: chemically bound particles), extremely preferably 3: 1-1: 3 (physically adsorbed particles: chemically bound particles), most preferably 3 1-1: 2 (physically adsorbed particles: chemically bonded particles). However, when the target antibody in the sample is measured, the content of the physically adsorbed particles in the contained liquid or, when the diluted liquid is used, in the mixed liquid of the contained liquid and the diluted liquid is 0.045% by mass or more. , There is a tendency that the improvement of the effect corresponding to the increase in the content of the particles is not recognized.

2. Measurement of antibody In the measurement method of the present invention, the antibody is measured using the above-mentioned solution of the present invention. That is, the containing solution of the present invention is brought into contact with a sample separated from a living body that can contain the target antibody, and the antigen antibody between the antigen carried on the insoluble carrier particles of the containing solution and the target antibody in the sample. The reaction of the fine particles due to the reaction can be detected. Although it is possible to carry out the measurement method of the present invention without using a diluent, it is preferably carried out using a diluent.

When the measurement is carried out using a diluted solution, the containing solution of the present invention diluted by mixing the diluted solution can be brought into contact with the sample, but preferably, the sample diluted by adding the diluted solution to the sample. And the liquid containing the present invention are brought into contact with each other. It is preferable to use a diluted solution having a volume ratio of 9 times or less, and it is particularly preferable to use a diluted solution having a volume ratio of 1 to 5 times or less with respect to the containing solution of the present invention.

As described above, the "sample isolated from the living body that can contain the target antibody" is not particularly limited as long as it may contain the antibody to be measured. For example, blood, serum, plasma, etc. Examples thereof include urine, lymph, puncture fluid , spinal fluid, sweat, saliva, gastric fluid, lung lavage fluid, feces and the like, of which blood, serum and plasma are preferable. Samples such as serum can be diluted with physiological saline or the like, if necessary. Samples diluted with physiological saline or the like are also included in "samples isolated from living organisms that may contain the target antibody". The dilution here is separate from the dilution with the diluent.

3. 3. Kit of the present invention The kit of the present invention is a measurement kit for carrying out the measurement method of the present invention, and includes the containing solution of the present invention and the following diluted solution as components.

Examples of the components in the kit of the present invention other than the containing liquid of the present invention include a standard product of the target antibody to be measured, a sample for quality control, a diluent and the like.

The diluent is preferably a liquid for diluting the "sample separated from the living body which may contain the target antibody" before contacting with the solution of the present invention, and the measurement of the target antibody is substantially performed. There is no particular limitation as long as it does not interfere with. As the diluent, a buffer solution is typical as in the case of the containing solution of the present invention. Examples of the buffer solution include Good's buffer solution such as HEPES buffer solution, TRIS buffer solution, glycine buffer solution, boric acid buffer solution and the like. If necessary, the diluent may contain, for example, various electrolytes, stabilizers, surfactants, sensitizers, and the like. The diluted solution is preferably the same as or similar to the solvent of the containing solution of the present invention.

Further, when the measurement method of the present invention is carried out using the kit of the present invention, the measurement may be carried out without using the diluent, but when the diluent is used, the containing liquid of the present invention is subjected to the measurement. A diluted solution of 9 times or less, particularly preferably 1 to 5 times by volume is used.

In the examples described later, the first reagent, which is a diluted solution before use, is the above-mentioned diluted solution, and the second reagent, which is a latex particle dispersion, is the containing solution of the present invention. One of the specific examples of the kit of the present invention is one containing these first reagent and second reagent as constituent elements.

Hereinafter, examples of the present invention will be described. % Is mass% unless otherwise specified.

[Manufacturing example]
1. 1. Latex The latex used in this example is as follows.
(1) Latex to be subjected to physical adsorption method-0.235 μm polystyrene latex particles (hereinafter referred to as physical latex 1)
0.223 μm polystyrene latex particles (hereinafter referred to as physical latex 2)
-0.310 μm polystyrene latex particles (hereinafter referred to as physical latex 3)

(2) Latex to be chemically bonded ・ 0.145 μm carboxylatex particles (hereinafter referred to as chemical latex 1)
-0.245 μm carboxylatex particles (hereinafter referred to as chemical latex 2)
0.300 μm carboxylatex particles (hereinafter referred to as chemical latex 3)

2. Preparation of supported antigen As the supported antigen derived from the bacteria of this example, the prepared antigen of Helicobacter pylori was used. The bacterial antigen was prepared according to the following procedure.

(1) Pre-culture The original strain was made into a slightly aerobic environment by Aneropack (Mitsubishi Gas Chemical Company, Inc.) using Poremedia (registered trademark) HP separation medium (Eiken Chemical Co., Ltd.) for selective separation of Helicobacter pylori. It was cultured at 37 ° C.

(2) Main culture As the culture solution for this culture, 200 mL of a culture solution obtained by adding 5% horse serum to Pearl Core (registered trademark) Brain Heart Infusion Bouillon medium'Eiken'(Eiken Chemical Co., Ltd.) was used. After catching a single colony of the above-mentioned preliminary medium in this main culture medium, McFarland No. The inoculated bacterial solution adjusted to 1.0 was inoculated, and the cells were cultured with shaking at 37 ° C. under slightly aerobic gas.

(3) Bacteriolysis After the above main culture is completed, the cells are collected by centrifugation, washed centrifugally with physiological saline, and the precipitate is lysed with a lytic solution (0.15 M NaCl, 0.1% polyoxyethylene octylphenyl ether, 5 mM). EDTA, 50 mM HEPES buffer containing 0.1% NaCl ₃ , pH 7.4) was added, and this was lysed by freeze-thaw.

(4) Preparation of antigen The above lysate of Helicobacter pylori was dialyzed against HEPES buffer (pH 7.4) containing _{0.15 M NaCl and 0.1% NaN 3, and then a centrifugal supernatant was obtained.} Was filtered through a membrane filter having a pore size of 0.45 μm. Using the filtered product as an antigen, antigen preparation was completed, and this was used as a "Helicobacter pylori lysate" and subjected to the following steps. The antigen protein concentration of Helicobacter pylori lysate was measured by the BCA method.

3. 3. Supporting step on latex particles (1) Supporting by physical adsorption method After adding 0.48 mL of 0.5 M CHES buffer (pH 8.8) to 21.1 mL of 0.09% polystyrene latex particle-containing solution, 8 mg / mL 0.25 mL of Helicobacter pyroli lysate was mixed. After stirring the mixed solution at room temperature for 30 minutes, 1.04 mL of a 10% BSA solution was added, and the total volume was adjusted to 24.0 mL with purified water. Further, after heat treatment at 56 ° C. for 4 hours, the unadsorbed antigen was removed by centrifugation. Latex particles from which unadsorbed antigens have been removed by centrifugation are redispersed in 4.0 mL of 0.01 M HEPES buffer (pH 7.4), 0.04 mL of 10% BSA solution is added, and then this is added at 56 ° C. for 2 Heat treated for hours. The heated antigen-adsorbing latex particle- containing liquid was filtered to prepare a physically adsorbed latex having a latex particle concentration of 0.48%.

The latex particle concentration of the physically adsorbed latex to be produced can be appropriately adjusted by appropriately changing the volume of 0.01 M HEPES buffer (pH 7.4) used for redispersion of latex particles after removing the unadsorbed antigen by centrifugation. Yes (same for chemical bonds).

(2) Support by chemical bonding method Add 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride to 6.4 mL of a 0.3% carboxylatex particle-containing liquid in an equal amount of 2 mol of carboxyl groups for 15 minutes. The carboxyl group was activated by a stirring reaction. After adding 63.2 mL of purified water and 1.28 mL of 0.5 M HEPES buffer (pH 7.0) to the latex particle- containing solution in which the carboxyl group has been activated, 0.340 mL of 8 mg / mL Helicobacter pylori lysate is added. It was mixed and reacted for 3 hours. 0.776 mL of a 10% BSA solution was added to the reaction mixture, and the mixture was reacted for 4 hours, and then the unadsorbed antigen was removed by centrifugation. Latex particles from which unbound antigens have been removed by centrifugation are redispersed in 4.0 mL of 0.01 M HEPES buffer (pH 7.4), 0.04 mL of 10% BSA solution is added, and heat treatment is performed at 56 ° C. for 4 hours. did. The heated antigen-bonded latex particle- containing liquid was filtered to prepare a chemically bonded latex having a latex particle concentration of 0.48%.

(3) Preparation of Latex Particle Dispersion Solution Prepared in 2.5 mL of HEPES buffer (pH 7.4) containing 0.3M NaCl, 0.8ML-arginine hydrochloride and 0.2% NaN _{3 according to (1) above.} Add 1.563 mL of the physically adsorbed latex and 0.521 mL of the chemically bonded latex prepared in (2) above, and add 0.15% of the physically adsorbed latex particles and 0.05% of the total amount to 5.0 mL with purified water. A latex particle dispersion containing chemically bonded latex particles was prepared.

Various mixing ratios can be obtained by appropriately selecting the addition amounts of the physically adsorbed latex and the chemically bonded latex.

4. Measurement system The measurement in this example was performed using the turbidity method.

The first reagent is a diluent (diluted solution before use), 0.05M HEPES, 0.15M NaCl, 0.5% chondroitin sulfate Na, 0.1% BSA, 0.1% PBC-34, 0. .1% NaCl ₃ pH 7.4, remaining amount is purified water.

The second reagent is a latex particle dispersion (containing solution of the present invention), 0.05M HEPES, 0.15M NaCl, 0.4M L-Arg HCl, 0.1% NaN ₃ , and latex particle pH 7. 4. The remaining amount is purified water.

The measurement was performed using an automatic biochemical analyzer JCA-BM2250 (JEOL Ltd.).

Specifically, in the apparatus, 60 μL of the first reagent was mixed with 6.0 μL of a sample (serum sample) diluted 5-fold with physiological saline, incubated at 37 ° C. for 5 minutes, and then the second reagent was added to this mixed solution. 20 μL of the reagent was mixed and reacted at 37 ° C., and the change in absorbance at two wavelengths of a main wavelength of 658 nm and a sub-wavelength of 805 nm was measured for about 3 minutes after mixing the second reagent. That is, in this example, the first reagent having a volume ratio of 3 times was used with respect to the second reagent (dilution ratio: 4 times).

As a reference method, measurement was performed by the ELISA method (E plate'Eiken'H. pylori II: Eiken Chemical Co., Ltd.).

[Example 1] Examination of mixing effect of two types of latex particles As described above, physically adsorbed latex particles (physical latex 1 ) having a particle size of 0.235 μm and carrying a helicobacter pyrori lysate by different methods, Reactivity of chemically bonded latex particles (chemical latex 1) having a particle size of 0.145 μm, when only physically adsorbed latex particles are used, when only chemically bonded latex particles are used, and when a mixture thereof is used. Was confirmed using a dilution series of pooled serum (Table 1, FIG. 1). The latex particle concentration of the second reagent used in the study was 0.15% when only physically adsorbed latex particles were used, 0.30% when only chemically bonded latex particles were used, and physical when a mixture was used. Adsorbed latex particles and chemically bonded latex particles were set to 0.15% and 0.30%, respectively. Serum samples were then measured to examine the specificity of each case (Table 2). The "unit of reactivity in Table 1" and "unit of the vertical axis in FIG. 1" are obtained by multiplying the amount of change in absorbance (ΔOD / min) by 10,000.

The concentrations of the physically adsorbed latex particles and the chemically bonded latex particles shown in Table 1 are the concentrations in the second reagent as described above, and the latex concentrations at the time of measurement are indicated by dilution with the first reagent, respectively. It is 1/4 of. For example, if "Physical Latex 1 0.15%" is displayed, the concentration of Physical Latex 1 at the time of measurement is "0.15 x 1/4 = 0.0375%".

By mixing the chemically bonded latex particles with the physically adsorbed latex particles , the positive matching rate is improved and the specificity is improved while maintaining a high negative matching rate, and the concentrations of the physically adsorbed latex particles and the chemically bonded latex particles in the second reagent. When were set to 0.15% and 0.30% (1: 2), respectively, favorable results were observed in the overall concordance rate (Table 2).

[Example 2] Examination of mixing ratio of two types of latex particles As described above, the first of physically adsorbed latex particles (physical latex 2) having a particle size of 0.223 μm, which carries a helicobacter pyrori lysate by different methods. 2 The concentration in the reagent was set to 0.20%, and chemically bonded latex particles (chemical latex 1) having a particle size of 0.145 μm were added so as to be 0-0.20%, and the serum sample was measured and peculiar. The sex was examined (Table 3).

Further, the relationship between the display of the concentrations of the physically adsorbed latex particles and the chemically bonded latex particles in Table 3 and the relationship between the concentrations at the time of measurement by 4-fold dilution with the first reagent is as described in Example 1.

When the chemically bonded latex particles were mixed, the matching rate was improved by increasing the mixing amount of the chemically bonded latex particles. In particular, as the concentration of the chemically bonded latex particles in the second reagent increased to 0%, 0.05%, 0.10%, 0.20%, the positive concordance rate increased to 53%, 67%, 73%, It was found that it was significantly improved to 80% and it was possible to avoid false negatives (missing). However, since the mixed amount of the physically adsorbed latex particles was 0.20%, which exceeded 0.18% (measured: 0.045%) described later, the increase in the concordance rate was suppressed (Table). 3).

[Example 3] Examination of mixing ratio of two types of latex particles (1)
As described above, the concentration of the physically adsorbed latex particles (physical latex 1) having a particle size of 0.235 μm in which the helicobacter pyrrol lysate was carried by different methods in the second reagent was set to 0.12%, and the particles were added to the particles. When chemically bonded latex particles (chemical latex 3) having a diameter of 0.300 μm were added so as to be 0-0.10%, the reactivity was determined to be LZ-H. The examination was carried out using the Helicobacter pylori antibody calibrator'Eiken'(Eiken Chemical Co., Ltd.) (Table 4, Fig. 2), and then the serum sample was measured to examine the specificity (Table 5). The "unit of reactivity in Table 4" and "unit of the vertical axis in FIG. 2" are obtained by multiplying the amount of change in absorbance (ΔOD / min) by 10,000.

Further, the relationship between the indication of the concentrations of the physically adsorbed latex particles and the chemically bonded latex particles in Tables 4 and 5 and their concentrations at the time of measurement by 4-fold dilution with the first reagent is as described in Example 1. ..

From this result, as the amount of the chemically bonded latex particles mixed with the physically adsorbed latex particles increased, the positive concordance rate improved, and false negatives (missing) could be avoided. Further, as the mixing amount of the chemically bonded latex particles is increased, the reactivity is improved, and the concentration ratio of the physically adsorbed latex particles and the chemically bonded latex particles in the second reagent is 12: 5-12: 10 (3: 1). Within -1: 3), the overall concordance rate was favorable (Table 5).

[Example 4] Examination of mixing ratio of two types of latex particles (2)
A study to the same effect as in Example 3 was carried out by further expanding the measurement range. The latex particles used are physical latex 1 and chemical latex 3 as in Example 3.

(1) The concentration of the physical latex 1 in the second reagent is fixed at 0.12%, and the concentration of the chemical latex 3 is 0% (physical adsorption: chemical bond = 7.5: 0) and 0.016% (physical adsorption: chemical bond = 7.5: 0). 7.5: 1), 0.048% (7.5: 3), 0.08% (7.5: 5), 0.12% (7.5: 7.5), 0.16% ( The specificity in the case of 7.5: 10) was examined by measuring a serum sample (Table 6). The H. pylori antibody level is LZ-H. It was obtained from the calibration curve prepared using the Helicobacter pylori antibody calibrator'Eiken'(Eiken Chemical Co., Ltd.).

Further, the relationship between the display of the concentrations of the physically adsorbed latex particles and the chemically bonded latex particles in Table 6 and the relationship between the concentrations at the time of measurement by 4-fold dilution with the first reagent is as described in Example 1.

(2) The concentration of the chemical latex 3 in the second reagent was fixed at 0.08%, and the concentration of the physical latex 1 was 0% (physical adsorption: chemical bond = 0: 5) and 0.024% (1. 5: 5), 0.072% (4.5: 5), 0.12% (7.5: 5), 0.18% (11.3: 5), 0.24% (15: 5) The specificity of the case was examined by measuring a serum sample (Table 7). The H. pylori antibody level is LZ-H. It was obtained from the calibration curve prepared using the Helicobacter pylori antibody calibrator'Eiken'(Eiken Chemical Co., Ltd.).

Further, the relationship between the display of the concentrations of the physically adsorbed latex particles and the chemically bonded latex particles in Table 7 and the relationship between the concentrations at the time of measurement by 4-fold dilution with the first reagent is as described in Example 1.

In the above (1) and (2), the effect of mixing the physically adsorbed latex particles and the chemically bonded latex particles in a wider range than that of Example 3 was confirmed.

That is, as a result of examining the concentration ratio of the physically adsorbed latex particles and the chemically bonded latex particles in the second reagent in the range of 7.5: 1-7.5: 10, in the example of 7.5: 1. , The effect of mixing these two types of latex particles was observed, and the overall concordance rate was favorable at the other 7.5: 3-7.5: 10 (within 3: 1-1: 3) (Table). 6).

Further, as a result of examining the above concentration ratio in the range of 1.5: 5-15: 5, 1.5: 5 (within 5: 1-1: 5) and 4.5: 5 and 7 At .5: 5 (within 3: 1-1: 3), the overall concordance ratio was favorable, but the concentration of the physically adsorbed latex particles in the second reagent was 0.18% (measured: 0.045%). ) In the above two cases (11.3: 5 and 15: 5 cases), although the compounding ratio itself was in the range of 3: 1-1: 3, the effect of improving the specificity by mixing was hindered (Table). 7).

In the case of using either the physically adsorbed latex particles or the chemically bonded latex particles , the difference between the positive concordance rate and the negative concordance rate was even more extreme, but this was due to the small number of samples. It is thought that there is.

[Example 5] Examination of particle size of latex particles As described above, a second reagent of physically adsorbed latex particles (physical latex 1) having a particle size of 0.235 μm, which carries a helicobacter pyrori lysate in a different manner. The medium concentration is 0.15%, and chemically bonded latex particles having a particle size of 0.245 μm or 0.300 μm (chemical latex 2 (medium particle size) and chemical latex 3 (large particle size), respectively) are added to this. % Mixing and each specificity was examined by measuring serum samples. The H. pylori antibody level is LZ-H. It was obtained from the calibration curve prepared using the Helicobacter pylori antibody calibrator'Eiken'(Eiken Chemical Co., Ltd.). As a result, the overall concordance rate (combined positive concordance rate and negative concordance rate) was 92% and 88%, respectively, and no substantial difference was observed between the two.

Furthermore, in order to confirm the effect on the measured value of the particle size between the physically adsorbed latex particles , the physical latex 2 (particle size: 0.223 μm (medium particle size)) and the physical latex 3 (particle size: 0.310 μm (large particle size)) The specificity of each of the diameters)) was measured at a concentration of 0.10% in the second reagent by the same method as described above, but the positive agreement rate, the negative agreement rate, and the overall agreement rate were substantially the same. No particular difference was observed.
Also in this Example 5, the relationship between the display of the concentrations of the physically adsorbed latex particles and the chemically bonded latex particles and the concentration at the time of measurement by 4-fold dilution with the first reagent is as described in Example 1. be.

From this result, the particle size of the chemically bonded latex particles and the particle size of the physically adsorbed latex particles both substantially affect the measurement specificity of the sample separated from the living body, at least in medium to large particle sizes. It was confirmed that there was no such thing.

Claims (11)

A liquid containing insoluble carrier particles containing both an insoluble carrier particle carrying a predetermined antigen by physical adsorption and an insoluble carrier particle carrying the antigen by a chemical bond can be obtained from a living body capable of containing a target antibody. A method for measuring an antibody, which comprises contacting with a separated sample to detect an aggregation reaction of the carrier particles due to an antigen-antibody reaction between an antigen carried on the carrier particles and a target antibody in the sample. The antibody measuring method according to claim 1, wherein the predetermined antigen is a mixture of a plurality of substances. The antibody measuring method according to claim 2, wherein the mixture of the plurality of substances is a lysate derived from a microorganism. The antibody measuring method according to any one of claims 1-3, wherein the predetermined antigen contains at least one substance having a molecular weight of 5000 or more. The antibody measuring method according to any one of claims 1-4, wherein the insoluble carrier particles are latex particles. An antibody measuring reagent for detecting the reaction of an antigen-supporting carrier particle by an antigen-antibody reaction with a target antibody in a sample separated from a living body, and an insoluble carrier particle in which a predetermined antigen is supported by physical adsorption. A reagent for measuring an antibody, which is a liquid containing insoluble carrier particles containing both an insoluble carrier particle in which the antigen is supported by a chemical bond. The measuring reagent according to claim 6, wherein the predetermined antigen is a mixture of a plurality of substances. The reagent for measurement according to claim 7, wherein the mixture of the plurality of substances is a lysate derived from a microorganism. The measuring reagent according to any one of claims 6-8, wherein the predetermined antigen contains at least one substance having a molecular weight of 5000 or more. The measuring reagent according to any one of claims 6-9, wherein the insoluble carrier particles are latex particles. A measurement kit comprising the measurement reagent and diluent according to any one of claims 6-10.

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